Among some of the earliest written records of man is an awareness that persons who recover from certain diseases cannot contract them again a second time. In today's terminology, such persons have become immune via a remarkably versatile set of adaptive processes which respond to an immense variety of infectious agents. Immune responses are encountered only in living vertebrates; and such immune responses constitute the principal means of defense against infection by pathogenic microorganisms.
In today's state of knowledge and technology, the infectious agents and the substances presented, produced or released by infectious agents are typically called "antigens". An almost limitless variety of macromolecules can behave as antigens--virtually all proteins; many polysaccharides; nucleoproteins, lipoproteins, and numerous synthetic polypeptides; and many small molecules if they are suitably linked to proteins or to synthetic polypeptides. Classically, an antigen has two properties: immunogenicity--the capacity to stimulate the formation of the corresponding antibodies and/or immune cells; and selectivity--the ability to react specifically with these antibodies or cells. Antigens are also distinct and different from "haptens" which, by definition, are not themselves immunogenic, but do react specifically with the appropriate corresponding antibodies or immune cells.
The term "immunogen" is often used for a substance or composition that stimulates the formation of the corresponding antibody in an organism able to respond. It is clear, however, that immunogenicity itself is not an intrinsic or inherent property of an infectious agent or a macromolecule. To the contrary, immunogenicity is dependent on the system and conditions employed in the introduction of the antigen into the body. One cardinal rule and condition is that the putative immunogen must be somehow recognized as alien, or foreign, or at least not as itself by the responding host.
In addition, for a variety of different public health reasons and medical reasons, man has employed immunogens and many different immunization procedures to increase active in-vivo resistance to infectious agents and to the products of pathogens. This has led to the ever-increasing study and development in the field of a unique problem: how to make and use an effective vaccine. By definition, a vaccine is a preparation used for immunization in which a suspension of infectious agents, some parts of them, or synthetic analogs of them, is given to a living subject in advance of a clinically apparent condition to establish active resistance to an infection or disease. The prevention of clinical infections and pathological disease states via the use of vaccines is considered one of the most effective and available procedures to combat illness. Merely representative of the range and diversity of vaccines available today to prevent infectious disease in man are those listed by Table 1 below.
TABLE 1 ______________________________________ Vaccines Preventing Infectious Disease in Man* Disease Immunogen ______________________________________ Diptheria purified diptheria toxoid Tetanus purified tetanus toxoid Smallpox infectious (attenuated) virus Yellow fever infectious (attenuated) virus Measles infectious (attenuated) virus Mumps infectious (attenuated) virus Rubella infectious (attenuated) virus Poliomyelitis infectious (attentuated) virus or inactivated virus Influenza inactivated virus Rabies inactivated virus Typhus fever killed rickettsiae Rickettsia prowazeki Typhoid and killed bacteria paratyphoid fevers Salmonelta typhi, S. schottmulleri, and S. paratyphi Pertussis killed bacteria Bordetella pertussis Cholera crude fraction of cholera vibrios Plague crude fraction of plague bacillus Tuberculosis infectious (attenuated) mycobacteria (bacille Calmette-Guerin of "BCG") Meningitis purified polysaccharide from Neisseria meningitidis Pneumonia purified polysaccharides from Streptococcus pneumoniae ______________________________________ *Microbiology, [Davis, Dulbecco, Eisen & Ginsberg, editors], Harper & Row 1988, p. 448.
Unfortunately, the development of vaccines and vaccination procedures which are effective against microbial antigens and infectious agents is a laborious and almost entirely empiric process. There are very few general rules which are reliable; and even these generalities are meager because they often do not apply uniformly or consistently. Among these are: that the material be antigenic--that is, that the composition contain chemical groupings which are not present in the living recipient and will become accessible to immunologically competent cells of the recipient which is to be immunized. In addition, it is essential that the material employed as a vaccine should have a sufficiently great molecular weight; in general, the larger a molecule is, the greater chance it will have of comprising foreign determinant groups on its surface. Also, it is often desirable that the substances in a vaccine be aggregated or be adsorbed on alum or other gels because these are usually more effective than soluble materials. The aggregated immunogens, by binding more effectively to cells in the living body, and by engaging more cell surface molecules on the specialized cells involved in generating immune responses, are often more stimulatory than dispersed or solute molecules; and the relatively slow rate of desorption from gels or emulsions maintains the antigen in tissues for longer periods of time. There also are variances and conditions regarding systemic versus local immunization procedures--the route of administration and the choice of site for injection being usually determined by convenience, but in some instances being limited by the very nature of the infectious agent, or vaccine efficacy itself, or by the nature or localization of the immune response desired. Finally, the number of administrations or injections of the immunogen used as a vaccine may vary markedly, varying commonly from month-long intervals to responses which last for years or even decades after a single immunization.
Owing to the major differences in the efficacy and usefulness of vaccines generally and to the risks involved in using live attenuated pathogens as vaccines, major research and development efforts have been directed towards the making of synthetic compositions of matter which would provide more effective immunizations and be more readily available for use as vaccines. Merely representative of the more recent innovations in this art are U.S. Pat. Nos. 5,219,577; 5,462,750; 4,251,509; 4,613,500; 5,206,015; 4,744,983; 4,657,762; 4,225,581; 4,329,332; 4,744,760; 4,501,726; 4,904,479; and the different publications cited within each of these issued patents.
In particular also, in any composition which is suitable for use as a vaccine or immunogen, it is essential that the conformational integrity and immunogenic/antigenic sites or "epitopes" of the proteins, macromolecules, or other agents be preserved intact. Changes in the structural configuration, structure, or spatial orientation of these molecules and compounds may and often does result in partial or total loss of antigenic activity and utility. Such changes in configuration are often caused by changing the environment surrounding or containing the compound or agent. Furthermore, the size and the ability of any associated carrier particle to minimize undesireable biological reactions of the recipient subject and to facilitate interaction of the compound with the immune system, are primary concerns when the composition or substance is used under in-vivo conditions. All of these factors must be taken into account when preparing a composition as a conjugate which is to be used as an immunogen and/or as a vaccine or as biomaterial for recognizing specific receptors.
Nevertheless, alterations in spatial orientation or structural alignment, physical denaturation, and other disruptive stereochemical or physical events often do destroy or markedly reduce the efficacy and value of an immunogen and conjugated compositions which have been intentionally prepared for use as a vaccine. Improvements in controlling orientation, overall configuration of the three-dimensional structure and overall size for a substance or prepared conjugate composition are thus of continuing importance and a current major concern in this art. Accordingly, methods and procedures by which such immunogens may be prepared in a chemically controlled manner and in a fixed spatial orientation and alignment are therefore deemed to be most advantageous and beneficial.